calpain and Leukemia--Erythroblastic--Acute

Hyperkinetic Jak2 tyrosine kinase signaling has been implicated in several human diseases including leukemia, lymphoma, myeloma, and the myeloproliferative neoplasms. Using structure-based virtual screening, we previously identified a novel Jak2 inhibitor named G6. We showed that G6 specifically inhibits Jak2 kinase activity and suppresses Jak2-mediated cellular proliferation. To elucidate the molecular and biochemical mechanisms by which G6 inhibits Jak2-mediated cellular proliferation, we treated Jak2-V617F expressing human erythroleukemia (HEL) cells for 12 h with either vehicle control or 25 μM of the drug and compared protein expression profiles using two-dimensional gel electrophoresis. One differentially expressed protein identified by electrospray mass spectroscopy was the intermediate filament protein, vimentin. It was present in DMSO treated cells but absent in G6 treated cells. HEL cells treated with G6 showed both time- and dose-dependent cleavage of vimentin as well as a marked reorganization of vimentin intermediate filaments within intact cells. In a mouse model of Jak2-V617F mediated human erythroleukemia, G6 also decreased the levels of vimentin protein, in vivo. The G6-induced cleavage of vimentin was found to be Jak2-dependent and calpain-mediated. Furthermore, we found that intracellular calcium mobilization is essential and sufficient for the cleavage of vimentin. Finally, we show that the cleavage of vimentin intermediate filaments, per se, is sufficient to reduce HEL cell viability. Collectively, these results suggest that G6-induced inhibition of Jak2-mediated pathogenic cell growth is concomitant with the disruption of intracellular vimentin filaments. As such, this work describes a novel pathway for the targeting of Jak2-mediated pathological cell growth.

Topics: Animals; Calpain; Cell Death; Cell Line, Tumor; Humans; Janus Kinase 2; Leukemia, Erythroblastic, Acute; Mice; Mice, Inbred NOD; Mice, SCID; Protein Kinase Inhibitors; Spectrometry, Mass, Electrospray Ionization; Stilbenes; Vimentin

Differentiation of erythroid cells is regulated by cell signaling pathways including those that change the intracellular concentration of calcium. Calcium-dependent proteases have been shown previously to process and regulate the activity of specific transcription factors. We show here that the protein levels of upstream stimulatory factor (USF) increase during differentiation of murine erythroleukemia (MEL) cells. USF was subject to degradation by the Ca(2+)-dependent protease m-calpain in undifferentiated but not in differentiated MEL cells. Treatment of MEL cells with the specific calpain inhibitor calpeptin increased the levels of USF and strongly induced expression of the adult alpha- and beta-globin genes. The induction of globin gene expression was associated with an increase in the association of USF and RNA po ly mer ase II with regulatory elements of the beta-globin gene locus. Calpeptin also induced high level alpha- and beta-globin gene expression in primary CD71-positive erythroid progenitor cells. The combined data suggest that inhibition of calpain activity is required for erythroid differentiation-associated increase in globin gene expression.

Topics: Animals; Calpain; Cell Differentiation; Dipeptides; Enzyme Inhibitors; Erythroid Cells; Female; Gene Expression Regulation; Globins; Leukemia, Erythroblastic, Acute; Mice; Mice, Inbred C57BL; Murinae; Promoter Regions, Genetic; RNA Polymerase II; Upstream Stimulatory Factors

Ligation of the cell surface molecule CD44 by anti-CD44 monoclonal antibodies (mAbs) has been shown to induce cell differentiation, cell growth inhibition and in some cases, apoptosis in myeloid leukemic cells. We report, herein, that exposure of human erythroleukemic HEL cells to the anti-CD44 mAb A3D8 resulted in cell growth inhibition followed by caspase-independent apoptosis-like cell death. This process was associated with the disruption of mitochondrial membrane potential (Delta Psi m), the mitochondrial release of apoptosis-inducing factor (AIF), but not of cytochrome c, and the nuclear translocation of AIF. All these effects including cell death, loss of mitochondrial Delta Psi m and AIF release were blocked by pretreatment with the poly (ADP-ribose) polymerase inhibitor isoquinoline. A significant protection against cell death was also observed by using small interfering RNA for AIF. Moreover, we show that calpain protease was activated before the appearance of apoptosis, and that calpain inhibitors or transfection of calpain-siRNA decrease A3D8-induced cell death, and block AIF release. These data suggest that CD44 ligation triggers a novel caspase-independent cell death pathway via calpain-dependent AIF release in erythroleukemic HEL cells.

Topics: Antigens, CD; Apoptosis; Apoptosis Inducing Factor; Calpain; Caspases; Cell Cycle; Cell Death; Cell Line, Tumor; Cell Survival; Cytochromes c; DNA, Neoplasm; Electrophoresis, Gel, Pulsed-Field; Humans; Hyaluronan Receptors; Isoquinolines; Leukemia, Erythroblastic, Acute; Membrane Potentials; Mitochondrial Membranes; Poly(ADP-ribose) Polymerase Inhibitors; Protein Transport; RNA, Small Interfering; Transfection

As indicated by direct evidence, obtained by altering the cell-membrane permeability for Ca2+ in murine erythroleukaemia (MEL) cells, calpain is the triggering factor which connects fluctuations of the intracellular Ca2+ concentrations to the decay of protein kinase C (PKC), as well as to the kinetics of cell differentiation induced by hexamethylenebisacetamide. Cell exposure to verapamil caused a profound decrease in the rate of PKC down-regulation and a slower initial rate of accumulation of mature erythroid cells, whereas addition of the Ca2+ ionophore A23187 produced opposite effects. The high susceptibility of PKC-delta to calpain degradation, at concentrations of Ca2+ much lower than those required for degradation of the other PKC isoforms, may be explained by the finding that this kinase isoform is predominantly associated with the cell membrane. The different cellular localizations, as well as the different susceptibilities to calpain digestion, further support the hypothesis that in MEL cells the various PKC isoforms play distinct biological functions that are critical for the maintenance of the undifferentiated state of the cell and for its commitment to terminal erythroid differentiation.

Topics: Acetamides; Animals; Calcium; Calpain; Cell Differentiation; Cell Membrane; Cell Membrane Permeability; Down-Regulation; Enzyme Activation; Intracellular Fluid; Isoenzymes; Kinetics; Leukemia, Erythroblastic, Acute; Mice; Protein Kinase C; Signal Transduction; Translocation, Genetic; Tumor Cells, Cultured

Calpain has been identified as the intracellular proteinase that catalyzes the selective down-regulation of protein kinase C (PKC) isoforms, occurring in the early stages of commitment to terminal erythroid differentiation of murine erythroleukemia (MEL) cells induced by hexamethylenebisacetamide. This conclusion has been reached through direct experiments performed with two MEL cell clones, one characterized by a high and the other by a low rate of differentiation. In both cell types, introduction of an anti-calpain antibody resulted in a significant delay in the onset of down-regulation of PKC isoforms, and in an increase in the latent period that precedes differentiation. Both cell lines also displayed reduced rates of PKC decay and accumulation of mature erythroid cells. Furthermore, in the fast-responding clone, calpastatin, the natural calpain-inhibitor protein, was found to be almost completely absent, resulting in activation and expression of proteolytic activity of calpain even at micromolar concentrations of Ca2+, a condition not sufficient to trigger calpain activation in the slowly responding clone which contains high levels of calpastatin. The fast-responding MEL cell clone, enriched with calpastatin, displayed a lower rate of cell differentiation, with a kinetics almost identical to that observed following introduction of the anti-calpain antibody. It is proposed that Ca(2+)-dependent proteolysis plays a crucial role for the progress of MEL cell differentiation through the specific degradation of PKC isozymes.

Topics: Acetamides; Antibodies; Antineoplastic Agents; Calcium; Calcium-Binding Proteins; Calpain; Cell Differentiation; Cell Line; Erythrocytes; Humans; Isoenzymes; Kinetics; Leukemia, Erythroblastic, Acute; Protein Kinase C; Tumor Cells, Cultured

Murine erythroleukemia cells contain a single type of calpain classified, on the basis of its calcium requirement, as a type I proteinase. The enzyme is practically inactive at concentrations of calcium below 10 microM and expresses maximal activity in the presence of 0.12-0.15 mM Ca2+. The affinity for Ca2+ cannot be reduced by exposure of the enzyme to conditions known to promote autoproteolysis of calpain. Expression of catalytic activity at lower concentrations of Ca2+, is promoted by the interaction with phospholipid vesicles or plasma membranes. Conditions that promote activation of calpain, induce also the self-inactivation of the enzyme. During terminal differentiation of murine erythroleukemia cells induced by HMBA, the intracellular level of calpain activity undergoes significative reduction. Similar decrease in calpain activity progressively occurs during the loss of sensitivity to HMBA as a result of density growth arrest.

Topics: Acetamides; Animals; Calcium; Calpain; Cell Differentiation; Enzyme Activation; Hemoglobins; Kinetics; Leukemia, Erythroblastic, Acute; Mice; Tumor Cells, Cultured